Insertion of furin protease cleavage sites in membrane proteins and uses thereof

ABSTRACT

Cleavage site for the protease furin is inserted between domains of a membrane glycoprotein. Upon cleavage by furin in the trans-Golgi network, the protein is separated into individual membrane-free domain that retains its native conformation. This protocol can be used to produce virus membrane protein domains for structural analysis and for trials as vaccines.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 10/841,787, filed onMay 7, 2004, now U.S. Pat. No. 7,223,390 which claims the benefit ofprovisional application U.S. Ser. No. 60/469,126, filed on May 9, 2003,now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the study and uses ofmembrane glycoproteins. More specifically, the present inventionprovides a method of producing membrane-free membrane glycoproteinsmaintained in native conformation.

2. Description of the Related Art

Many membrane glycoproteins are assembled within cells in a highlyconstrained and energy rich conformation. The membrane proteins ofmembrane-containing viruses are examples of energy rich proteins. Theseproteins assemble in the endoplasmic reticulum (ER) throughintermediates which are stabilized by disulfide bonds. Because of thishigh energy configuration it is difficult if not impossible to maintainthese proteins in native conformation when extracting them from theirassociated membrane. Extracting these proteins from the membrane resultsin their collapse into a normative, relaxed configuration that makesstructural analysis on these proteins difficult. In the case of virusmembrane proteins, the normative conformation makes these proteinsineffective for use as subunit viral vaccines.

In the case of influenza virus, this conformation problem was overcomeby the discovery of an accessible protease site in the HA1-HA2 membraneglycoprotein (Wiley and Skehel, 1977). This site allowed the release ofthe protein ectodomain upon treatment of intact virus with the protease.The released ectodomain retained its native conformation, therebyallowing the determination of its structure at atomic resolution byX-ray crystallography (Wiley and Skehel, 1977).

Most membrane proteins, however, do not contain an accessible proteasesite such as that found in influenza virus. This fact and the failure ofother methods of protein purification have made it impossible to obtainthese proteins in native conformation. Thus, the prior art is deficientin a method of producing membrane-free membrane glycoproteins maintainedin native conformation. The present invention provides a solution tothis long-standing need and desire in the art.

SUMMARY OF THE INVENTION

The present invention provides a procedure of using naturally occurringcellular proteases to produce membrane protein domains that maintainnative conformation upon release from their membrane bilayers. Thisevent of proteolytic cleavage and release occurs after the protein hasbeen exported from the endoplasmic reticulum and thus after the processof disulfide bridge formation, folding and oligomerization with otherproteins (if necessary) occurs. After engaging the furin protease in theGolgi apparatus, the protein is converted to a nonmembrane-associatedspecies which can be purified from the growth media by protocols thatprevent loss of native conformation. This protocol provides newopportunities for the production of virus membrane protein domains forstructural analysis and for trials as vaccines.

Virus carrying the furin insertion can be grown to high titer in hostcells that do not express furin. These mutant viruses may be used asvaccines because when injected into mammalian host, these viruses wouldinfect and begin the process of assembly but “self destruct” at the laststage of protein assembly at the trans Golgi network.

Thus, in one embodiment, the present invention comprises a method ofproducing a membrane glycoprotein domain, wherein the domain ismembrane-free and is maintained in native conformation. This method maycomprise the steps of: inserting a furin cleavage sequence in a regionthat divides the glycoprotein into separate domains; expressing theglycoprotein in a host cell; cutting the glycoprotein by furin in thetrans-Golgi network of the host cell, thereby producing a membraneglycoprotein domain; secreting the glycoprotein domain from the hostcell; and purifying the glycoprotein domain from the culture medium ofthe host cell, wherein the domain is membrane-free and is maintained innative conformation.

In another embodiment, the present invention comprises a method ofproducing a domain of alphavirus membrane glycoprotein useful as asubunit vaccine candidate, wherein said domain is membrane-free and ismaintained in native conformation. This method generally comprises thesteps of: inserting a furin cleavage sequence in a region that dividesthe glycoprotein into separate domains; expressing the glycoprotein in ahost cell; cutting the glycoprotein by furin in the trans-Golgi networkof the host cell, thereby producing a membrane glycoprotein domain;secreting the glycoprotein domain from the host cell; and purifying theglycoprotein domain from the culture medium of the host cell, whereinthe domain is membrane-free and is maintained in native conformation.

In another embodiment, the present invention comprises a method ofproducing vaccine candidates for alphavirus. This method comprises thesteps of inserting a furin cleavage sequence in a region that divides amembrane glycoprotein of the alphavirus into separate domains;incorporating sequence encoding the membrane glycoprotein comprising thefurin cleavage sequence into a vector encoding the alphavirus;expressing the alphavirus in a host cell that does not express furin;and collecting alphaviruses produced by the host cell, wherein thecollected viruses are vaccine candidates for alphavirus.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Functional and structural domains in the E1 glycoprotein ofSindbis virus are separated by amino acids 129-140. Inserting furincleavage sites at residue 130, 133 or 139 would cleave the protein intothe 17 kD functional domain (which is released from the membrane) andthe structural domain (which is retained in the membrane). Sites at E1392 and 393 would release the entire ecto domain.

FIG. 2: Polyacrylamide gel electrophoresis of proteins produced bySindbis virus mutants containing furin cleavage sites in the E1glycoprotein. Numerical designation indicates the amino acid site in E1where cleavage should occur. Y420, wild-type virus; P75, non-virusmessenger RNA; E1, envelope protein 1; E2, envelope protein 2; C, capsidprotein.

FIG. 3: Polyacrylamide gel electrophoresis of proteins produced bySindbis virus mutants containing furin cleavage sites at position E1 393(F393). The proteins are designated E1, E2 for the two normal virusglycoproteins and E1* for the furin truncated E1 protein. C is capsidprotein. P75, a mutant containing a non-virus RNA; Y420, wild-typevirus.

DETAILED DESCRIPTION OF THE INVENTION

Furin is a protease which resides in the trans-Golgi network ofeukaryotic cells (Moehring et al., 1993). Its function is to cleaveproteins at a step just prior to their delivery to their final cellulardestination. Furin recognizes a consensus amino acid sequence, RXRR (SEQID NO. 1), RXRK (SEQ ID NO. 2) or KXKR (SEQ ID NO. 3) (where X is anyamino acid, Moehring et al., 1993) and cuts proteins which contain thesesequences when they reach the trans-Golgi network.

In the present invention, a furin cleavage site is introduced into anexposed (externally situated) domain of a membrane glycoprotein. Themodified protein will go through its normal process of folding andassembly to attain its native configuration. These events are requiredfor its export from the endoplasmic reticulum. After export from theendoplasmic reticulum, the protein travels along the secretory pathwayto reach the cell surface. When the protein reaches the trans-Golginetwork, it is cleaved by the furin protease. The proteolytic eventreleases the ectodomain of the protein from the membrane bilayer withoutcompromising its conformation. The protein is now a secreted protein andcan be purified from the surrounding media by an appropriatepurification protocol.

To demonstrate the feasibility of this process, the membraneglycoprotein E1 of the prototype alphavirus Sindbis virus was chosen asa model. The alphaviruses are representatives of a class of viruses(arboviruses) which are responsible for significant human disease suchas Dengue Fever, West Nile Fever, Venezuelan Encephalitis, Yellow Feveretc. There are over 600 of these agents known but only one effectivevaccine (against Yellow Fever) is currently available. Attempts toproduce vaccines by producing subunits of extracted virus proteins ordenatured virus have failed because of the loss of native proteinconformation as described above.

The E1 glycoprotein of Sindbis virus is assembled in the endoplasmicreticulum of virus-infected cells into a compact, highly constrained andenergy rich configuration. Correct folding is a prerequisite for itsexport from the endoplasmic reticulum to the cell surface. Attempts toremove the virus E1 protein from the membrane result in the loss ofnative conformation as disulfide bridges shuffle bringing the protein toa normative configuration.

It has been shown that a correctly folded form of this protein isdivided into two separate disulfide bridge stabilized domains and thatthe junction between these two domains is around amino acid E1-129(Mulvey and Brown, 1994) (FIG. 1). The first of these domains (aminoacids 1-129) contains the function of membrane penetration (functionaldomain), while the second domain (130-39.8) holds the icosahedrallattice intact (structural domain). Data presented below indicate thatinsertion of a furin cleavage site in the region separating thefunctional domain and the structural domain results in releasing thefunctional domain in native conformation from the membrane proteincomplex.

The choice of the sites where the furin cleavage site should be insertedcan be determined based on the structure of the protein or, in casewhere the structure is not available, biochemical and/or sequenceanalyses. In general, the sites should be within segments ofpredominantly polar and or charged residues, most likely to be on thesurface of the protein. If the three dimensional structure is known, theinsertion sites should be in surface loops connecting well-orderedsecondary structure elements with extensive hydrophobic interface.

When the structure of the protein is not available, hydrophobicity-basedmethods, secondary structure prediction methods and sequence alignmentof homologues can often help reveal such candidate sites. If a smallamount of the protein is available, limited proteolytic digestionfollowed by chromatographic co-fractionation and N-terminal polypeptidesequencing/mass spectroscopy can help determine such candidate siteswhich are accessible to protease digestion and nonessential for formingan integral protein structure. Cutting at such site would separate theprotein into individual domains.

The instant method of obtaining membrane-free membrane glycoprotein canbe applied to a number of viruses such as HIV, Herpes viruses,coronaviruses etc. In general, the furin cleavage site can be insertedinto any virus membrane protein if that virus can replicate in a CHOcell line deficient in the protease furin or in a furin defective cellline that supports replication of the virus. The released membrane-freeecto domain of the viral glycoprotein can be used as a subunit vaccine.

In another embodiment, high titer of virus particles carrying the furininsertion can be generated in furin negative mammalian host cells. Theseviruses can be potential vaccine candidates. When injected intomammalian host, these viruses would infect and begin the process ofassembly but “self destruct” at the last stage of protein assembly atthe trans Golgi network due to cleavage by furin. This approach wouldwork for many viruses (e.g. HIV, Herpes etc) whenever furin-minus celllines are available to support the growth of the mutant viruses.

As used herein, “membrane glycoprotein” refers to any integral membraneprotein which is assembled in the endoplasmic reticulum and delivered tofinal destination by a cellular route that passes through the transGolgi network.

As used herein, “membrane-free membrane glycoprotein” refers to anintegral membrane protein which has been released from its membrane bycutting the protein at some point in the ectodomain with a protease.

As used herein, “native conformation” refers to the conformationachieved by a protein as it is folded in the endoplasmic reticulum. Forviral protein, it also refers to the functional form exists in a matureinfectious virus.

The present invention is directed to a method of producing a membraneglycoprotein domain, wherein said domain is membrane-free and ismaintained in its native conformation. In one embodiment, this methodcan be used to produce a domain of a viral membrane glycoprotein such asan alphavirus membrane glycoprotein. The resulting viral membraneglycoprotein domain is useful as a subunit vaccine candidate.

First of all, a furin cleavage sequence is inserted in a region thatdivides the membrane glycoprotein into separate domains. In general,suitable regions include the surface of the protein, a surface loop or aregion with predominant polar residues. Preferably, the furin cleavagesequence is SEQ ID NOs. 1, 2 or 3. Upon expression of such modifiedglycoprotein in a host cell, the glycoprotein would be separated intodifferent domains by furin cleavage in the trans-Golgi network.Subsequently, the glycoprotein domains can be purified from the culturemedium of the host cell, wherein the purified domains are membrane-freeand are maintained in native conformation.

The present invention is also directed to a method of producing vaccinecandidates for alphavirus. The method involves inserting a furincleavage sequence in a region that would divide a membrane glycoproteinof alphavirus into separate domains. In general, suitable regionsinclude the surface of the protein, a surface loop or a region withpredominant polar residues. Preferably, the furin cleavage sequence isSEQ ID NOs. 1, 2 or 3 or a fragment or obvious variant of one of thesefurin cleavage sequences. The modified glycoprotein is then incorporatedinto a vector encoding the alphavirus and expressed in host cells thatdo not express furin. Alphaviruses produced by these host cells would bevaccine candidates for alphavirus.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

EXAMPLE 1

Release of Protein Domain in Native Conformation after Furin Cleavage

To release the first domain of the E1 glycoprotein of Sindbis virus fromthe membrane protein complex, a furin protease cleavage site wasinserted in the region separating the functional domain from thestructural domain of the protein. This was done using the Quick-Change™technique of mutagenesis (Stratagene) in a full length cDNA clone of thevirus RNA (Rice et al., 1987).

The primers used to produce these mutations are shown in Table 1.Mutations were made to place the furin sensitive sequence at positionsE1-130 (RXRK, SEQ ID NO.2), E1-133 (KXKR, SEQ ID NO.3) and E1-139 (RXRR,SEQ ID NO.1) using the naturally occurring amino acid sequences whenpossible. Selection of these sites was based on research whichdemonstrates that these sites would be exposed on the protein surfaceand thus would be available for protease cleavage (Phinney et al., 2000;Phinney and Brown, 2000).

It is predicted that these mutations would result in the release of thedistal E1 protein domain from the E1 protein upon exposure to the cellassociated enzyme furin. This would change the molecular weight of theintact E1 protein from a molecular weight of 58 kD to two proteins ofmolecular weights approximately 17 kD and 41 kD. To control for theeffects of amino acid changes on the normal folding of the virusglycoproteins, the mutation-containing virus RNAs were transfected intoa CHO (Chinese Hamster Ovary) cell line which does not have the furinprotease (CHO-RPE40) (Moehring et al., 1993; Moehring and Moehring,1983).

FIG. 2 shows proteins produced in mammalian cells transfected withconstructs for the E1 mutants F130, F133 and F139. Placement of thecleavage site at positions E1-130 and E1-133 resulted in the productionof new protein species migrating at molecular weights of approximately41 and 17 kD as predicted. The amount of the 17 kD protein wasrelatively small as the proteins shown were those which were associatedwith the cell and it is likely that most of the 17 kD protein wassecreted into the media. These proteins were not seen in the wild typetransfection (Y420) or in cells transfected with a non-virus message(P75).

E1 393 mutant was intended to release the entire E1 ectodomain (see FIG.1). The SDS PAGE of proteins immunoprecipitated from the media of BHKcells transfected with the RNA of the 393 mutant are shown in FIG. 3.

As was the case with mutant E1 139, mutation at E1 392 failed to producethe desired phenotype (data not shown). The transfection of BHK cellswith RNA produced from the cDNA clone of mutant furin E1 392 resulted inthe release into the media a protein migrating faster than glycoprotein.E2 and which was immunoprecipitated by antibody against the whole virus.Wild-type E1 has 439 amino acids (58 kDa), wild-type E2 has 423 aminoacids (53 kDa), and the truncated E1 ectodomain is predicted to have 392amino acids (51 kDa), having lost 47 amino acids from the carboxylterminus.

As shown in FIG. 3, E1 393 mutant (F393) produced more truncated E1 thanwild-type E1, indicating that there was efficient processing at the 393cleavage site.

TABLE 1 Primer Sets For The Production of E1 Furin Sensitive MutationsSEQ ID Mutant Primer set NO. F-130 Sense 5′GCACACTCGCGCGCGGAAAGTAGG 3′ 4Antisense 5′CCTACTTTCCGCGCGCGAGTGTGC 3′ 5 F-133 Sense5′CCGCGATGAAAGTAAAACGCCGTATTGTGTACG 3′ 6 Antisense5′CCGTACTCAATACGGCGTTTTACTTTCATGCGG 3′ 7 F-139 Sense5′CTACGGGAGGACTAGGAGATTCCTAGATGTGT 3′ 8 Antisense5′ACACATCTAGGAATCTCCTAGTCCTCCCGTAC 3′ 9 F-392 Sense5′GAGCACCCCGAGACACAAAAGAGACCAAGAATTTC 3′ 10 Antisense5′GAAATTCTTGGTCTCTTTTGTGTCTCGGGGTGCTC 3′ 11 F-393 Sense5′GAGCACCCCGCACAGAAATAGACGAGAATTTCAAGC 3′ 12 Antisense5′GCCGGCTTGAAATTCTCGTCTATTTCTGTGCGGGGT 3′ 13

EXAMPLE 2

Replication of Viruses Carrying the Furin Insertion

The effects of furin protease site insertions on the production ofinfectious virus are shown in Table 2. Table 2 shows that mutantscontaining the furin cleavage site produce very low levels of infectiousvirus when their RNA is transfected into the furin protease containingBHK-21 cells.

In contrast, these mutants produce wild-type (Y420) amounts of viruswhen transfected into CHO-RPE40 cells that do not have furin activity.For the mutants F-130 and F-133, this result shows that the presence ofthe furin cleavage site at these locations does not prevent correctfolding E1 as it is incorporated into infectious virus. Infectiousviruses production is inhibited by 5-6 orders of magnitude in the BHKcell because furin has cut the folded E1 protein into two separatedomains. The mutant F-139 shows a similar inhibition of growth in BHKcells even though significant cleavage of E1 does not take place. In thecase of the 139 substitution, the location of this mutation eliminatesone of two glycosylation sites (Pletnev et al., 2001) as evidenced bythe faster migration of this partially glycosylated protein. Thatelimination of glycosylation site may lead to conformational change thatprevents infectious virus production.

The E1 393 mutant produced a titer of 10³ virions from BHK cellscompared to a titer of 10⁹ produced by wild-type virus under similarconditions of RNA transfection. Mutant 393 produced 10⁷ to 10⁸virions/ml in CHO RPE-40 cells (furin negative cells). Thus, the E1 393mutant had significantly less virus production than wild-type or E1 130and E1 133 mutants (Table 2). The reason for this difference is notclear but may imply a reduced efficiency of folding or oligomerformation in the furin 393-substituted glycoprotein.

TABLE 2 Replication of E1 Furin Mutants Growth in CHO- Mutant Growth inBHK-21 RPE40 Y420 (wild type) 1.0 × 10⁹ 1.25 × 10¹⁰ F 130 4.0 × 10⁴ 5.23× 10¹⁰ F 133 4.4 × 10⁵ 5.13 × 10¹⁰ F 139 5.8 × 10⁴ 1.95 × 10¹⁰ F 393 10³10⁷-10⁸

The following references were cited herein:

-   Moehring et al., Expression of mouse furin in a Chinese hamster cell    resistant to Pseudomonas exotoxin A and viruses complements the    genetic lesion. J Biol. Chem. 268:2590-4 (1993).-   Moehring and Moehring, Strains of CHO-K1 cells resistant to    Pseudomonas exotoxin A and cross-resistant to diphtheria toxin and    viruses. Infect. Immun. 41:998-1009 (1983).-   Mulvey and Brown, Formation and rearrangement of disulfide bonds    during maturation of the Sindbis virus E1 glycoprotein. J. Virol.    68:805-812 (1994).-   Phinney and Brown, Sindbis virus glycoprotein E1 is divided into two    discrete domains at amino acid 129 by disulfide bridge    connections. J. Virol. 74:9313-6 (2000).-   Phinney et al., The surface conformation of Sindbis virus    glycoproteins E1 and E2 at neutral and low pH, as determined by mass    spectrometry-based mapping. J. Virol. 74:5667-78 (2000).-   Pletnev et al., Locations of carbohydrate sites on alphavirus    glycoproteins show that E1 forms an icosahedral scaffold. Cell    105:127-36 (2001).-   Rice et al., Production of infectious RNA transcripts from Sindbis    virus cDNA clones: mapping of lethal mutations, rescue of a    temperature-sensitive marker, and in vitro mutagenesis to generate    defined mutants. J. Virol. 61:3809-19 (1987).-   Wiley and Skehel, Crystallization and x-ray diffraction studies on    the haemagglutinin glycoprotein from the membrane of influenza    virus. J Mol. Biol. 112:343-7 (1977).

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference.

1. A recombinant virus comprising a non-native membrane glycoprotein,the glycoprotein comprising an inserted furin cleavage sequence thatprovides a furin cleavage site in the glycoprotein that is non-native tosaid glycoprotein, such that furin cleavage of the glycoprotein releasesan ectodomain of said glycoprotein from the membrane.
 2. The virus ofclaim 1, wherein the inserted furin cleavage sequence is RXRR (SEQ IDNO. 1).
 3. The virus of claim 1, wherein the inserted furin cleavagesequence is RXRK (SEQ ID NO. 2).
 4. The virus of claim 1, wherein theinserted furin cleavage sequence is KXKR (SEQ ID NO. 3).
 5. The virus ofclaim 1, wherein the inserted furin cleavage sequence is introduced intoan exposed domain of said membrane glycoprotein.
 6. The virus of claim1, wherein the inserted furin cleavage site is in a surface loop.
 7. Thevirus of claim 1, wherein the membrane glycoprotein is an arbovirusmembrane glycoprotein.
 8. The virus of claim 1, wherein the membraneglycoprotein is a HIV or Herpes membrane glycoprotein.